This invention relates to the use of part of tetanus toxin for delivering a composition to the central nervous system of a human or animal. This invention also relates to a hybrid fragment of tezanus toxin, a polynucleotide that hybridizes with natural tezanus toxin, and a composition containing the tetanus toxin fragment as an active molecule. Further, this invention relates to a vector comprising a-promoter and a nucleic acid sequence encoding the tetanus toxin fragment.
Tetanus toxin is produced by Clostridium tetani as an inactive, single, polypeptide chain of 150 kD composed of three 50 kD domains connected by protease-sensitive loops. The toxin is activated upon selective proteolytic cleavage, which generates two disulfide-linked chains: L (light, 50 kD) and H (heavy, 100 kD) [Montecucco C. and Schiavo G. Q. Rev. Biophys., (1995), 28: 423-472].
Evidence for the retrograde axonal transport of tetanus toxin to central nervous system (CNS) has been described by Erdmann et al. [Naunyn Schmiedebergs Arch Phamacol., (1975), 290:357-373], Price et al. [Science, (1975), 188:945-94], and Stoeckel et al. [Brain Res., (1975), 99:1-16]. In each of these studies, radiolabeled toxin was found inside membrane bound vesicles. Another property was the transynaptic movement of tetanus toxin that was demonstrated first by autoradiographic localization of 125 I-labeled tetanus toxin in spinal cord linterneurons after injection into a muscle [Schwab and Thoenen, Brain res., (1976), 105:218-227].
The structure of this tetanus toxin has been elucidated by helting et al. [J.Biol. Chem., (1977), 252:187-193]. Papain cleaves the tetanus toxin in two fragments:                the C terminal part of the heavy chain, 451 amino acids, also called fragment C; and        the other part contained the complementary portion called fragment B linked to the light chain (fragment A) via a disulfide bond.        
European Patent No. EP 0 030 496 B1 showed the retrograde transport of a fragment B-IIb to the CNS and was detected after injection in the median muscle of the eye in primary and second order neurons. This fragment may consist of “isofragments” obtained by clostridial proteolysis. Later, this fragment B-IIb was demonstrated to be identical to fragment C obtained by papain digestion by Eisel et al. [EMBO J., 1986, 5:2495-2502].
This EP patent also demonstrated the retrograde transport of a conjugate consisting of a Ibc tetanus toxin fragment coupled by a disulfide bond to B-IIb from axonal endings within the muscle to the motoneuronal perikarya and pericellular spaces. (The Ibc fragment corresponds to the other part obtained by papain digestion as described above by Helting et al.). There is no evidence that this conjugate was found in second order neurons. The authors indicated that a conjugate consisting of the fragment B-IIb coupled by a disulfide bond to a therapeutic agent was capable of specific fixation to gangliosides and synaptic membranes. No result showed any retrograde axonal transport or a transynaptic transport for such conjugate.
Another European Patent, No. EPD 0 057 140 B1, showed equally the retrograde transport of a fragment IIc to the CNS. As in the European Patent No. EP 0 030 496 31, the authors indicated that a conjugate consisting of the fragment IIc and a therapeutic agent was capable of specific fixation, but no result illustrated such allegation. This fragment IIc corresponds to the now called fragment C obtained by papain digestion.
Francis et al. [J. Biol. Chem., (1995), 270(25):15434-15442] just led an in vitro study showing the internalization by neurons of hybrid between SOD-1 (Cu Zn superoxide dismutase) and a recombinant C tetanus toxin fragment by genetic recombination. This recombinant C tetanus toxin fragment was obtained from Halpern group. (See ref. 11).
Moreover, Kuypers H. G. J. M and Ugolini G. [TINS, (1990), 13(2):71-75] indicated in their publication concerning viruses as transneuronal tracers that, despite the fact that tetanus toxin fragment binds to specific receptors on neuronal membranes, transneuronal labeling is relatively weak and can be detected only in some of the synaptically connected neurons.
Notwithstanding these advances in the art, there still exists a need for methods for delivering compositions into the human or animal central nervous system. There also exists a need in the art for biological agents that can achieve this result.